Studies on mammalian nuclear architecture aim to understand how 2 meters of DNA is folded into a nucleus of 10 μm across, while allowing accurate expression of the genes that specify the cell-type, and how this is faithfully propagated during each cell cycle. Progress to in this field has largely come from microscopy studies, which revealed that genomes are non-randomly arranged in the nuclear space. For example, densely packed heterochromatin is separated from more open euchromatin and chromosomes occupy distincy territories in the nuclear space. An intricate relationship exists between nuclear positioning and transcriptional activity. Although transcription occurs through the nuclear interior, active genes that cluster on chromosomes preferentially locate at the edge or outside of their chromosome territory. Individual genes may migrate upon changes in their transcription status, as measured against relatively large nuclear landmarks such as chromosome territories, centromeres or the nuclear periphery. Moreover, actively transcribed genes tens of megabases apart on the chromosome can come together in the nucleus, as demonstrated recently by fluorescence in situ hybridization (FISH) for the β-globin locus and a few, selected, other genes. Besides transcription, genomic organization is associated with the coordination of replication, recombination and the probability of loci to translocate (which can lead to malignancies) and the setting and resetting of epigenetic programs. Based on these observations it is thought that the architectural organization of DNA in the cell nucleus is a key contributor of genomic function.
Different assays have been developed to allow an insight into the spatial organization of genomic loci in vivo. One assay, called RNA-TRAP has been developed (Carter et al. (2002) Nat. Genet. 32, 623) which involves targeting of horseradish peroxidase (HRP) to nascent RNA transcripts, followed by quantitation of HRP-catalysed biotin deposition on chromatin nearby.
Another assay that has been developed is called chromosome conformation capture (3C) technology, which provides a tool to study the structural organisation of a genomic region. 3C technology involves quantitative PCR-analysis of cross-linking frequencies between two given DNA restriction fragments, which gives a measure of their proximity in the nuclear space (see FIG. 1). Originally developed to analyse the conformation of chromosomes in yeast (Dekker et al., 2002), this technology has been adapted to investigate the relationship between gene expression and chromatin folding at intricate mammalian gene clusters (see, for example, Tolhuis et al., 2002; Palstra et al., 2003; and Drissen et al., 2004). Briefly, 3C technology involves in vivo formaldehyde cross-linking of cells and nuclear digestion of chromatin with a restriction enzyme, followed by ligation of DNA fragments that were cross-linked into one complex. Ligation products are then quantified by PCR. The PCR amplification step requires the knowledge of the sequence information for each of the DNA fragments that are to be amplified. Thus, 3C technology provides a measure of interaction frequencies between selected DNA fragments.
There is an important need for high-throughput technology that can systematically screen the whole genome in an unbiased manner for DNA loci that contact each other in the nuclear space.
The present invention seeks to provide improvements in 3C technology.